Identification and evaluation of novel anchoring proteins for cell surface display on Saccharomyces cerevisiae
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The development of arming yeast strains as whole-cell biocatalysts involves a selection of effective anchoring proteins to display enzymes and proteins on yeast cell surface. To screen for novel anchoring proteins with improved efficiency, a bioinformatics pipeline for the identification of glycosylphosphatidylinositol-anchored cell wall proteins (GPI-CWPs) suitable for attaching passenger proteins to the cell surface of Saccharomyces cerevisiae has been developed. Here, the C-terminal sequences (CTSs) of putative GPI-CWPs were selected based on the criteria that the sequence must contain a serine/threonine-rich (S/T) region of at least 30% S/T content, a total threonine content of at least 10%, a continuous S/T stretch of at least 130 amino acids in length, and a continuous T-rich region of at least 10 amino acids in length. Of the predicted 790 proteins, 37 putative GPI-CWPs were selected from different yeast and fungal species to be evaluated for their performance in displaying yeast-enhanced green fluorescent protein and β-glucosidase enzyme. This led to the identification of five novel anchoring proteins with higher performance compared to α-agglutinin used as benchmark. In particular, the CTS of uncharacterized protein in Kluyveromyces lactis, namely 6_Kl, is the most efficient anchoring protein of the group. The CTS of 6_Kl protein provided a β-glucosidase activity of up to 23.5 U/g cell dry weight, which is 2.8 times higher than that of the CTS of α-agglutinin. These identified CTSs could be potential novel anchoring protein candidates for construction of efficient arming yeasts for biotechnology applications in the future.
KeywordsCell surface display Anchoring protein GPI-anchored protein Saccharomyces cerevisiae β-Glucosidase Bioinformatics
We thank Dr. Wananit Wimuttisuk and Mr. Christopher Keith Campbell for critically reading the manuscript.
This work was financially supported by the National Center for Genetic Engineering and Biotechnology, Thailand (Grant no. P1300748).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with animals or human participants performed by any of the authors.
- Dujon B, Sherman D, Fischer G, Durrens P, Casaregola S, Lafontaine I, de Montigny J, Marck C, Neuvéglise C, Talla E, Goffard N, Frangeul L, Aigle M, Anthouard V, Babour A, Barbe V, Barnay S, Blanchin S, Beckerich J-M, Beyne E, Bleykasten C, Boisram A, Boyer J, Cattolico L, Confanioleri F, de Daruvar A, Despons L, Fabre E, Fairhead C, Ferry-Dumazet H, Groppi A, Hantraye F, Hennequin C, Jauniaux N, Joyet P, Kachouri R, Kerrest A, Koszul R, Lemaire M, Lesur I, Ma L, Muller H, Nicaud J-M, Nikolski M, Oztas S, Ozier-Kalogeropoulos O, Pellenz S, Potier S, Richard G-F, Straub M-L, Suleau A, Swennen D, Tekaia F, Wésolowski-Louvel M, Westhof E, Wirth B, Zeniou-Meyer M, Zivanovic I, Bolotin-Fukuhara M, Thierry A, Bouchier C, Caudron B, Scarpelli C, Gaillardin C, Weissenbach J, Wincker P, Souciet J-L (2004) Genome evolution in yeasts. Nature 430:35–44CrossRefGoogle Scholar
- Eisenhaber B, Schneider G, Wildpaner M, Eisenhaber F (2004) A sensitive predictor for potential GPI lipid modification sites in fungal protein sequences and its application to genome-wide studies for Aspergillus nidulans, Candida albicans, Neurospora crassa, Saccharomyces cerevisiae and Schizosaccharomyces pombe. J Mol Biol 337:243–253CrossRefGoogle Scholar
- Hamada K, Terashima H, Arisawa M, Yabuki N, Kitada K (1999) Amino acid residues in the ω -minus region participate in cellular localization of yeast glycosylphosphatidylinositol-attached proteins. J Bacteriol 181:3886–3889Google Scholar
- Phillips GJ (2001) Green fluorescent protein--a bright idea for the study of bacterial protein localization. FEMS Microbiol Lett 204:9–18Google Scholar
- Sambrook J, Russel DW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
- Shibasaki S, Ueda M, Iizuka T, Hirayama M, Ikeda Y, Kamasawa N, Osumi M, Tanaka A (2001) Quantitative evaluation of the enhanced green fluorescent protein displayed on the cell surface of Saccharomyces cerevisiae by the fluorometric and confocal laser scanning microscopic analyses. Appl Microbiol Biotechnol 55:471–475CrossRefGoogle Scholar
- Van der Vaart JM, te Biesebeke R, Chapman JW, Toschka HY, Klis FM, Verrips CT (1997) Comparison of cell wall proteins of Saccharomyces cerevisiae as anchors for cell surface expression of heterologous proteins. Appl Environ Microbiol 63:615–620Google Scholar